Nucleic acids are linear polymers consisting of individual nucleotide subunits which are covalently linked together via phosphodiester bonds. Oligonucleotides are now widely used in the biomedical field as nucleic acid sequencing primers, diagnostic probes and modulators of gene function. One of the most promising uses of oligonucleotides is in the field of antisense therapeutics.
Oligonucleotides can be conveniently synthesized using enzymatic or chemical methods, with the latter generally providing for larger scale production than the former. One of the most widely used methods of chemically synthesizing oligonucleotides is based on phosphoramidite solid-phase chemistry (See Methods in Molecular Biology, Volume 20: Protocols for Oligonucleotides and Analogs, S. Agrawal editor, Humana Press Inc., Totowa, N.J., 437-463 (1993)) Now fully automated, this method can be used to chemically produce oligonucleotides in commercial quantities. (See Oligonucleotides and Analogues: A Practical Approach. Edited by F. Eckstein, I.R.L. Press, Oxford, England, 1-24 (1991).)
Despite the convenience of chemical synthesis, the number of steps and the harshness of the chemicals involved leads to the formation of a product which may contain toxic chemical impurities, such as damaged nucleotide bases. Because the level of impurities is normally relatively low, chemically synthesized oligonucleotides are still suitable for use in many different applications. However, certain applications require a product which is substantially free of chemical impurities. In particular, when the application involves a biological system, the presence of chemical impurities can have a deleterious effect. Moreover, when the application involves an in vivo therapeutic agent, chemical purity is essential. Enzymatic synthesis, which can be used to produce an oligonucleotide product in an aqueous solution that is essentially free of toxic chemical impurities and hazardous byproducts is thus preferred for these applications.
The use of oligonucleotides in biological systems is also compromised by the presence of nucleases which catalyze the breakdown of nucleic acids by hydrolysis of phosphodiester bonds (See The Biochemistry of the Nucleic Acids: Chapter 4, Degradation and Modification of Nucleic Acids, Roger L. P. Adams et al., Chapman & Hall, London, England, 97-108 (1992)). Such a breakdown can cause a significant reduction in the biological activity of oligonucleotides in vivo thus resulting in diminished therapeutic effectiveness. This degradation can be controlled by modifying or substituting the phosphodiester bonds with a more nuclease-resistant analog, such as phosphotriester, phosphorothioate or methylphosphonate.
Phosphorothioate-containing oligonucleotides efficiently resist degradation by many nucleases, and are thus preferred for use in some biological systems. Phosphorothioate linkages have a sulfur in place of oxygen as one of the non-bridging atoms bonded to phosphorous. This substitution produces chirality at the phosphorous which is designated as having either the Rp or Sp diastereomer orientation. Since the chiral orientation is an important factor which influences duplex structure, enzyme recognition, conformation and/or hybridization kinetics, it is desirable to use chirally pure phosphorothioate-containing oligonucleotides. Other modified oligonucleotides such as those containing phosphotriester and methylphosphonate linkages also contain a substitution of one of the oxygen atoms bonded to phosphorous and thus exist as diastereomers.
Chemical synthesis of phosphorothioate-containing oligonucleotides generally lacks stereoselectivity and results in the formation of a product which is a heterogeneous mixture of two different chiral species. Attempts to stereochemically control the synthesis of chirally pure oligonucleotides have met with mixed success (Stec, et al., Nucleic Acids Research, 19(21): 5883-5888 (1991)). Cook (U.S. Pat. No. 5,212,295) has described the chemical synthesis of modified oligonucleotides with greater than 75% chiral purity. In comparison, enzymatic synthesis of phosphorothioate-containing oligonucleotides can be used to produce chirally pure product, since several polymerases form only phosphorothioate linkages having the Rp orientation (Eckstein, Ann. Rev. Biochem. 54:367-402 (1985)).
The enzymatic synthesis of other modified oligonucleotides is also well known in the art. Methylphosphonate-containing oligonucleotides can be produced enzymatically using DNA polymerases .alpha. and .epsilon. from human placenta, DNA polymerase .beta. from rat liver, and reverse transcriptases from HIV and arian myeloblastosis virus (Dyatkina, et al., Nucleic Acids Research Symposium Series 24: 238 (1991)).
The economical enzymatic synthesis of any oligonucleotide, whether modified or not, depends on the ability to efficiently utilize the components of the synthesis reaction to form a product which is sufficiently pure for its intended use. In some instances, it is desirable to use synthesis components which are capable of functioning repeatedly and are thus "reusable". For example, Richards et al. (PCT WO 92/05287) describes the use of reusable synthesis templates which function repeatedly in the same synthesis reaction. In other instances, it may be desirable to use synthesis components which function only once in a synthesis reaction and are thereafter degraded or rendered nonfunctioning. Walder, et al. (European Patent Application 496,483 A2) describe the use of RNA-containing primers that are cleaved in order to prevent the formation of undesired amplification products in subsequent synthesis reactions.
Even though many of the prior art methods are suitable for use in the enzymatic synthesis of oligonucleotides, large scale synthesis of chirally pure oligonucleotides has yet to be optimized. It is therefore an object of the present invention to provide for the economical synthesis of oligonucleotides which are chirally pure.
Regardless of whether phosphorothioate-containing oligonucleotides are chemically or enzymatically synthesized, their resistance to hydrolysis makes them difficult to cleave as is sometimes desirable during synthesis and/or purification. Most studies involving phosphorothioate-containing oligonucleotides and restriction endonucleases have centered on the design of oligonucleotides which are protected from cleavage (Nakamaye, et al., Nucleic Acid Research 14(24): 9679-9699 (1986); Richards et al., supra; and Vosberg, et al., Journal of Biol. Chem., 257(11): 6595-6599(1982)).
The present invention involves the identification of several restriction endonuclease recognition sequences ("restriction sequences") which are capable of being recognized and cleaved by restriction endonucleases, even when present in fully phosphorothioated oligonucleotides. In the enzymatic synthesis of phosphorothioate oligonucleotides, utilizing templates which direct the synthesis of products containing these restriction sequences facilitates separation and purification of oligonucleotides. In addition, the present invention provides a general method of cleaving phosphorothioate oligonucleotides which is useful in other basic molecular biology procedures.
None of the references herein are admitted to be prior art.